Almost all mRNA present in eukaryotic cells terminates in a sequence of approximately 20 to 250 adenosine nucleotides, a fact that is used in most mRNA purification methods. Today, a standard method for the detection and quantification of mRNA is the combined use of reverse transcriptase (RT) and PCR (RT-PCR) (Larrick, J. W., Trends Biotechnol. 10, 146-152 (1992); Kawasaki, E. S., PCR Protocols: A Guide to Methods and Applications (eds. Innis, M. A. et al.), Academic Press, San Diego, Calif. (1990)). The initial material for this method is purified total RNA (rRNA, tRNA, mRNA) or purified mRNA. The latter is used because the enzymes, particularly polymerases, used in the RT-PCR are easily inhibited by contamination.
The purification of RNA and mRNA is known from several methods including extraction with organic solvents (e.g. phenol/chloroform), purification via oligo(dT)-cellulose, or isolation with the aid of oligo(dT)-coated magnetic particles (Sambrook, J., Fritsch, E. F. and T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2nd edition (1989); Farrell, R. E., RNA Modologies: A Laboratory Guide for isolation and Characterization, Academic Press (1993)). These methods are complex and difficult to automate.
Purification of mRNA can also be achieved via hybridization of the poly(A)-carrying 3'-end of the eukaryotic mRNA, using biotinylated oligo(dT). Hybrids of biotin-oligo(dT) and mRNA are then bound to avidin or streptavidin-coated magnetic particles and isolated. This method has a drawback in that only large amounts of mRNA hybrid can be isolated; the mixture is, hence, not sensitive enough for the detection of specific mRNAs.
An assay using RT-PCR on solid phase, such as magnetic particles, was then developed to address this problem. A drawback of this method is, however, that after binding and washing of the mRNA, the magnetic particles must be transferred into reaction vessels that are suitable for PCR. These vessels must be especially stable to heat, in view of the elevated temperatures at which PCR is carried out. This requires another separation step. Each added step in a reaction compromises it further.